X-linked adrenal hypoplasia congenita (X-linked AHC) is a disorder of adrenal gland development. AHC manifests as the abnormal development of the adult zone of the adrenal cortex and results in low serum concentrations of glucocorticoids, mineralocorticoids and androgens. AHC typically presents early in infancy, although occasionally this disease will present in children and adults. If untreated, adrenal insufficiency is rapidly lethal as a result of acidosis, hypoglycemia, and shock. The AHC locus was originally mapped to the Xp21 region of the X chromosome. This region was later identified to contain the DAX-1 gene (Dosage sensitive sex-reversal, adrenal hypoplasia congenita on the X chromosome, gene 1). DAX-1 is a member of the large family of proteins called the nuclear hormone receptors. Specifically, DAX-1 is classified as an orphan receptor, due to the lack of identification of a cognate ligand that binds to DAX-1. DAX-1 has been shown to interact with other members of the nuclear hormone receptor family such as estrogen receptor (ER), androgen receptor (AR), thyroid hormone receptor (TR), and steroidogenic factor-1 (SF-1). The result of this interaction ultimately ends in transcriptional repression. DAX-1 has been shown to bind to several DNA sequences and repress transcription, however, very little is known of the proteins that interact with DAX-1 in order to carry out transcriptional repression. To date, only three DAX-1 cofactor proteins have been identified. However, whether these cofactor proteins interact with DAX-1 specifically in human adrenal gland tissue and are important in mediating the AHC phenotype together with DAX-1 is unknown. The proposed study will investigate both known as well as novel protein cofactor proteins of DAX-1 in the adrenal gland. We will a) examine expression of known DAX-1 cofactor proteins for expression in human adrenal gland, b) investigate the effect of DAX-1 AHC mutations on the interaction with known cofactor proteins, and c) isolate and identify proteins novel DAX-1 cofactor proteins in human adrenal cells. The experiments detailed in this proposal will provide a better understanding of the complex of proteins that function together with DAX-1 in order to mediate the AHC phenotype. The research described in this proposal will lead to future studies that will continue to explore the molecular determinants of DAX-1 that ultimately lead to X-linked AHC. PUBLIC HEALTH RELEVANCE: The adrenal glands are vital organs that produce and release hormones that regulate metabolism, excretion, response to stress, and sexual development in humans. Disorders that result in the abolishment of proper development of the adrenal glands, such as Adrenal Hypoplasia Congenita (AHC), are lethal if left untreated. Examination of the key factors that function in the adrenal will further our understanding of not only how these factors produce these devastating diseases when they are disrupted, but also how they regulate appropriate development of the adrenal glands when they are intact.